SNP Genotyping Support—Getting Started
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TaqMan® SNP Genotyping Assays
The probe labels are provided in the assay documentation and also online. Simply look up the assay by typing the assay ID into the search bar on lifetechnologies.com. Click on the “View Details” button, and under the Product Details look for the Context Sequence. The assays are always in a [VIC®/FAM™ dye] order. From the context sequence, you can see which base is first (thus labeled with VIC® dye), and which is second (thus labeled with FAM™ dye).
No. TaqMan® SNP assays contain competitive probe sequences with a 1 bp mismatch where the SNP is located. The other probe will still bind at a lower efficiency, producing some signal for the other fluorophore. Therefore, these are not quantitative.
We recommend diluting the 40X and 80X TaqMan® SNP Genotyping Assays to a 20X working stock with 1X TE buffer. The 1X TE buffer should be 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, and be made using DNase-free, sterile-filtered water.
You can use our Custom Assay Design Tool to submit your SNP sequence for an assay design. Alternatively, you can also use Primer Express® Software to design an assay. The DNA sequence needs to have at least 30 bases on each side of the SNP site. The more sequence you provide, the greater chance you have of the tool creating a passing design. The ideal sequence length is ~ 200 bases on either side of the SNP site. For the best-performing assay, we recommend prescreening the sequence for other SNPs, repeats, and regions of low complexity. These variations should be masked prior to submitting the sequence. This will prevent the design tool from designing the primers or probe over those regions, which could reduce the efficiency of the assay if they were used. For more details on how to screen and mask the sequence, please follow the guidelines here.
Reaction Setup and Sample Preparation
We recommend using ~1–20 ng of good-quality gDNA per well. The gDNA should have an A260/A280 ratio of ~1.7–1.9. It is also important to try to add the same amount of gDNA to every well.
We recommend using either the TaqMan® Universal Master Mix (I or II) or the TaqMan® Genotyping Master Mix. The TaqMan® Genotyping Master Mix has the advantage of proven performance with up to 3 days of pre- and post-PCR stability, allowing you to set up plates ahead of time or read the plates later (see the data here).
We do not provide control samples. However, Coriell has a large gDNA repository and you may be able to find controls there. Go to: http://ccr.coriell.org/ and choose “SNP Search”. Enter your rs number or gene name. If there are samples available you will see them in the table, such as in this example below. Browse the different genotypes for your desired controls.
If a control is not available, you can have one synthesized using GeneArt® Gene Synthesis.
SymphoniqPCR™ Analysis Software for Genotyping Analysis
Yes, any user with a Lifetechnologies.com account can access the SymphoniqPCR™ Analysis Software.
SymphoniqPCR™ Analysis Software allows analysis of more experiment files, contains more flexibility in analysis with enhanced file organization.
The software only analyzes data from Life Technologies™ qPCR instruments.
Storage plans for purchase will be announced shortly.
This is roughly 140 OpenArray® plates.
Please see the benefits below:
- Allows access from anywhere using web-based browser
- Enables flexibility of analysis and advanced quality control features
- Bigger project sizes for multi plate analysis
QuantStudio™ 12K Flex, QuantStudio™ 6 and 7, ViiA™ 7, 7500 Fast (eds files only), 7500, 7900 HT, and StepOne™ and StepOnePlus ™ Real-Time PCR Systems are compatible with SymphoniqPCR™ Analysis Software.
Data can be combined into a single project, however data must be analyzed separately via the use of analysis groups.
.las files cannot be imported into SymphoniqPCR™ Analysis Software.
Downloading is not available, but sharing of files and projects is possible.
Analysis Groups is a feature to divide your data based on various groups (like different instruments, for example) and apply specific analysis settings. As data is analyzed, there is a dropdown that can be selected for each analysis group.
Yes, only genotyping .sds files can be analyzed by the genotyping analysis module. Gene expression files from 7500 v1.4 files are not validated.
Here are the system requirements:
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- Google® Chrome™ Browser Version 21 or later
- Microsoft® Internet Explorer® Browser Version 10 or later
- Mozilla® Firefox® Browser Version v10.0.12 or later
- Windows® XP, Vista, 7, or 8
- Macintosh® OS 8 or later
An internet connection capable of 300kbps/300kbps (upload/download) or better. If your network employs a firewall that restricts outbound traffic, it must be configured to allow outbound access to apps.lifetechnologies.com on HTTPS-443.
For Research Use Only. Not for use in diagnostic procedures.